Skin constitutes the largest organ of the human body, serving as a protective barrier against physical injury, radiation and temperature. Skin consists of an underlying mesenchymal (dermal) layer and an outer epithelial (epidermal) layer.
Skin wound healing is regulated by various mechanisms including cell-cell interactions, extracellular matrix production and a number of cytokines and growth factors (see Paul Martin. Wound Healing—Aiming for Perfect Skin Regeneration. Science 276 (75) 1997). Important aims of wound treatment include rapid wound closure and a functionally and aesthetically satisfactory scar.
Wound healing in skin proceeds via an overlapping pattern of events including coagulation, inflammation, tissue formation (epithelialization, formation of granulation tissue, matrix) and tissue remodeling. PDGF BB is implicated in the processes of inflammation, granulation, tissue formation, re-epithelization, matrix formation and remodeling. The process of repair is mediated in large part by interacting molecular signals, primarily cytokines. Initial injury triggers coagulation and an acute local inflammatory response followed by mesenchymal stem cell recruitment, proliferation and matrix synthesis. Failure to resolve the inflammation can lead to chronic non healing wounds, whereas uncontrolled matrix accumulation, can lead to excess scarring.
One of the major growth factors known to be crucial for wound healing is platelet-derived growth factor (PDGF), a family of heparin-binding proteins made by many types of cell, including platelets, macrophages, smooth muscle and endothelial cells. The PDGF-BB (homodimer of PDGF-B) variant is the only growth factor approved by the Food and Drug Administration (FDA) for the treatment of non-healing diabetic ulcers. However, excessive usage of PDGF-BB has been linked to an increased risk of cancer.
Glycosaminoglycans are complex, linear, highly charged carbohydrates that interact with a wide range of proteins to regulate their function; they are usually synthesized attached to core protein. GAGs are classified into nonsulfated (HA) and sulfated (CS, DS, KS, heparin and HS).
Among the GAGs, the heparan sulfate (HS) family is of particular interest because of its ability to interact with targeted proteins based on specific sequences within its domains. The family (heparin and HS) consist of repeating uronic acid-(1→4)-D-glucosamine disaccharide subunits with variable pattern of N-, and O-sulfation. For example, the anti-coagulant activity of heparin requires 30-sulfation in glucosamine residue with a unique pentasaccharide arrangement (Lindahl U, Backstrom G, Hook M, Thunberg L, Fransson L A, Linker A. Structure of the antithrombin-binding site in heparin Proc Natl Acad Sci USA. 1979; 76:3198-202.). A unique sulfation pattern is also apparent for ECM proteins; an avid heparin-binding variant that binds FN is particularly highly charged, with 7 to 8 N-sulfated disaccharides being required, and with a larger domain than usual (>14 residues) (Falcone D J, Salisbury B G J. Fibronectin stimulates macrophage uptake of low-density lipoprotein-heparin-collagen complexes Arteriosclerosis. 1988; 8:263-73; Mahalingam Y, Gallagher J T, Couchman J R. Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin require heparan sulfate with specific properties. J Biol Chem. 2007; 282:3221-30). However, HS differs from such sulfated heparins by having highly sulfated NS domains separated by unsulfated NA domains; such dispositions provide unique arrangements for selectively binding proteins, without the side effects of heparin (Gandhi N S, Mancera R L. The Structure of Glycosaminoglycans and their Interactions with Proteins. Chem Biol Drug Des. 2008; 72:455-82.).
The disaccharide composition of HS can be elucidated through a series of enzymatic cleavages (Venkataraman G, Shriver Z, Raman R, Sasisekharan R. Sequencing complex polysaccharides. Science. 1999; 286:537-42; Desai U R, Wang H M, Linhardt R J. Specificity studies on the heparin lyases from Flavobacterium-heparinum Biochemistry. 1993; 32:8140-5; Shriver Z, Sundaram M, Venkataraman G, Fareed J, Linhardt R, Biemann K, et al. Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin. Proc Natl Acad Sci USA. 2000; 97:10365-70) using the Flavobacterium heparinium enzymes heparinase I, II and III to cleave the glycosidic bonds. More than 90% depolymerization of heparin or HS is possible when all 3 heparinases are used in combination (Karamanos N K, Vanky P, Tzanakakis G N, Tsegenidis T, Hjerpe A. Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate. J Chromatogr A. 1997; 765:169-79; Vynios D H, Karamanos N K, Tsiganos C P. Advances in analysis of glycosaminoglycans: its application for the assessment of physiological and pathological states of connective tissues. J Chromatogr B. 2002; 781:21-38.). The resulting disaccharide mixtures can be analyzed by PAGE (Hampson I N, Gallagher J T. Separation of radiolabeled glycosaminoglycan oligosaccharides by polyacrylamide-gel electrophoresis Biochem J. 1984; 221:697-705), SAX-H PLC (Skidmore M A A, Yates E and Turnbull J E. Labelling heparan sulfate saccharides with chromophore, fluorescence and mass tag for HPLC and MS separations. Methods in Molecular biology. 2009; 534:157-69), or highly sensitive capillary electrophoresis (CE) (Lamari F, Militsopoulou M, Gioldassi X, Karamanos N K. Capillary electrophoresis: a superior miniaturized tool for analysis of the mono-, di-, and oligosaccharide constituents of glycan moieties in proteoglycans. Fresenius J Anal Chem. 2001; 371:157-67; Karamanos N K, Vanky P, Tzanakakis G N, Hjerpe A. High performance capillary electrophoresis method to characterize heparin and heparan sulfate disaccharides. Electrophoresis. 1996; 17:391-5; Sudhalter J, Folkman J, Svahn C M, Bergendal K, Damore P A. Importance of size, sulfation, and anticoagulant activity in the potentiation of acidic fibroblast growth-factor by heparin J Biol Chem. 1989; 264:6892-7; Militsopoulou M, Lamari F N, Hjerpe A, Karamanos N K. Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection. Electrophoresis. 2002; 23:1104-9) by comparison to known disaccharides standards.